B. Materials and Methods
4. Northern blot
4.3 Amplification of a cDNA-Northern blot probe
4.3.1 Preparation of competent cells
Competent bacteria are able to incorporate external DNA. Treatment with calcium chloride and rubidium chloride is one method to initiate this uptake.
E. coli DH5α were cultivated in LB-medium (20 ml) at 37°C up to an optical density (OD600 nm) of 0.3 to 0.5. Cells were centrifuged (5,000 rpm, 5 min, 4°C), the pellet was resuspended in 1 ml of ice cold solution A (3-[N-Morpholino]propansulfonic acid (MOPS) pH 7.0, 10 mM; rubidium chloride 10 mM), added up to 10 ml solution A and centrifuged again. The supernatant was discarded, the pellet resuspended in 1 ml solution B (MOPS, pH 6.5, 100 mM, calcium chloride 50 mM, rubidium chloride 10 mM) and after adding up to 10 ml with solution B, incubated on ice for 30 min. After centrifugation and gentle resuspensation in solution B, 10% glycerol was added and competent cells were storable at –70°C for several weeks.
4.3.2 Transformation of competent E. coli
Transformation of E. coli, the uptake of free external DNA into bacteria cells, was first described by Mandel and Higa (Mandel et al., 1970).
100 µl of competent bacteria suspension were thawed on ice and incubated with 0.1 µg plasmid DNA for 30 min. The suspension was incubated at 42°C for 60 sec and transferred into 1 ml LB-medium. The mixture was again incubated at 37°C for 1 h, mixed and 20 µl or 200 µl, were plated onto LB-agar-ampicillin plates. The successfully transformed bacteria were able to grow overnight at 37°C in single colonies.
Materials and Methods 37
4.3.3 Mini-prep DNA isolation
Success of transformation was controlled by picking single colonies of bacteria grown on LB agar ampicillin plates and propagating them. The plasmids were isolated by lysing the bacteria with lysozyme, Triton X-100 (non-ionic detergent), and heat. Chromosomal DNA thereby clings to the cell membrane and can be separated together with cell debris from plasmid DNA by centrifugation. The solved plasmid DNA is then precipitated by isopropanol and washed with ethanol 70% (v/v) to remove in isopropanol insoluble salts.
The identification of the plasmid DNA takes place by gel electrophoresis.
STET
Sucrose 8% (m/v) Triton X-100 5% (m/v)
Tris-HCl pH 8.0 50 mM
EDTA 50 mM
One single bacteria colony was incubated in a sterile 15 ml centrifugation tube in 2 ml LB/ampicillin overnight under vigorous shaking (300 rpm, 37°C) in a Unihood 550 incubator (Uniequip, Martinsried, Germany). The next morning, 500 µl of the bacteria suspension were transferred to a sterile reaction tube and stored at 4 °C. The remaining bacteria suspension was centrifuged (100 x g, 5 min) and the supernatant removed. The bacteria pellet was resuspended in 200 µl STET. By addition of 16 µl lysozyme solution (10 mg/ml, sodium salt) and incubation at 75°C (60 sec) the bacteria were lysed.
Centrifugation at 12,000 x g for 10 min separated the bacteria DNA (in the pellet) from the plasmid DNA (in the supernatant). Plasmid DNA was precipitated by addition of isopropanol 50% (v/v) (5min, RT) and after centrifugation at 12,000 x g for 10 min the pellet was washed with ethanol 70% (v/v). After a final centrifugation step (12,000 x g, 3 min), the pellet was air dried and dissolved in 40 µl TE buffer and stored at –20°C until electrophoresis.
4.3.4 Agarose gel electrophoresis
Plasmid DNA was identified by electrophoresis in an 1% (m/v) agarose gel in 1x TBE-buffer. 12 µl DNA solution were mixed with 3 µl commercial loading buffer (Promega, Heidelberg, Germany) and separated in 1x TBE electrophoresis buffer in a horizontal electrophoresis chamber at 80 V. 0.5 µg of known plasmid served as control.
10x TBE (Tris borate EDTA)
Tris 900 mM Boric acid 889 mM
38 Northern blot
EDTA 25 mM
4.3.5 Midi-prep DNA isolation with Qiagen Plasmid Midi Kit
Larger amounts of highly purified DNA for the preparation of a DNA Northern blot probe were isolated with the Quiagen Midi Kit (Quiagen GmbH, Hilden, Germany). This kit is based on the principle of alkaline lysis with consecutive chromatographic purification via anionic exchange columns. Thereby, DNA is separated from polysaccharides, RNA, and other contaminants.
Bacteria were lysed with NaOH/SDS and proteins and DNA were denatured. The method is based on the fact that by neutralization with high concentrations of potassium acetate the relatively low molecular plasmid DNA again renatures, while proteins, DNA, and cell debris remain bound to salt/SDS complexes and can be separated by centrifugation. The following purification by anionic exchange columns removes salt- and protein impurities.
By addition of isopropanol to the eluate the plasmid DNA is precipitated.
Buffer P1
Tris-HCl pH 8.0 50 mM
EDTA 10 mM
RNase A 100 µg/ml
Buffer QBT
NaCl 750 mM
MOPS pH 7.0 50 mM
Ethanol 15% (v/v) Triton X-100 0.15% (m/v)
Buffer QC
NaCl 1.0 M
MOPS pH 7.0 50 mM
Ethanol 15% (v/v)
Buffer QF
NaCl 1.25 M
Tris-HCl pH 8.5 50 mM
Ethanol 15% (v/v)
STE (Sodium chloride Tris EDTA buffer)
NaCl 100 mM
Tris-HCl pH 8.0 10 mM
EDTA 1 mM
Materials and Methods 39
The successfully transformed E. coli DH5α bacteria were cultivated in 50 ml LB/ampicillin to an optical density (600 nm) of 1 to 1.5 and centrifuged at 1,000 x g at 4°C for 15 min.
The pellet was resuspended in 10 ml of buffer P1 and lysed with 4 ml 200 nM NaOH/1%
(m/v) SDS (5 min, RT) followed by incubation on ice for 15 min with 4 ml ice cold 3 M potassium acetate pH 5.5. The pellet was separated by centrifugation (20,000 x g, 30 min, 4°C) and the supernatant was cleared completely by centrifugation once more for 15 min.
The supernatant was placed on a equilibrated Quiagen column (Quiagen-tip 100, with 4 ml QBT buffer) and the column was washed twice with 10 ml buffer QC. The eluate was discarded and the DNA bound to the column eluted with 5 ml buffer QF. The DNA in the eluate was precipitated with 0.7 volumes of isopropanol. The resulting pellet after centrifugation at 15,000 x g (4°C, 30 min) was washed with 70% (v/v) ethanol and air dried. After resolving in H2O, concentration of DNA was determined according to 3.2.
4.3.6 Restriction endonuclease digestion
To obtain the inserted HO-1 DNA sequence for preparation of the radioactive probe the purified circular plasmid was cut with the restriction enzymes Eco RI and Hind III (see 4.2.1).
Reaction mixture
Plasmid DNA 20 µg Restriction enzyme 3 µl
Buffer 10 µl
H2O ad 100 µl
The reaction mixture was prepared on ice and incubated for 5 h at 37°C. To separate the restriction products an agarose gel electrophoresis was performed. The respective products were identified by comparison with a DNA ladder mix (GeneRuler DNA Ladder Mix, MBI Fermentas, St. Leon Roth, Germany).
4.3.7 Extraction of DNA from agarose gels
DNA fragments were extracted and purified from agarose gels with the QIAquick gel extraction kit from Qiagen (Hilden, Germany). Test principle is the adsorption of nucleic acids to a silica-gel membrane in presence of high salt concentration and a defined pH.
Contaminants such as salts, enzymes, unincorporated nucleotides, agarose and dyes do not bind to the silica-gel membrane but flow through the column.
To extract the HO-1 fragment, the corresponding lane was cut out of the agarose gel and
40 Northern blot
3 volumes of buffer QG was added to 1 volume of gel slice. The gel was solubilized by incubating the mixture at 50°C for 10 min. The yellow color of the solution indicated the correct pH value (<7.5). One volume of isopropanol was added and the sample was mixed. To bind DNA to a QIAquick spin column, the sample was applied to a column in a collection tube and was centrifuged for 1 min at 14,000 rpm. The flow through was discarded and after addition of 0.5 ml of buffer QG the column was centrifuged again.
After washing with 0.75 ml of buffer PE the flow through was removed and the column was centrifuged for 1 min to separate residual ethanol. To elute the DNA from the silica-gel membrane the column was placed in a clean reaction tube and 50 µl of H2O were added to the column before centrifugation. Under these conditions (pH 7-8.5 and low salt) maximum elution efficiency is achieved. Until further investigation the DNA was stored at –20°C.
4.3.8 Sequencing of double stranded DNA
The generation of the correct plasmid insert was confirmed by sequencing the isolated product by the company Toplab, Martinsried, Germany. Sequencing by the method of Sanger et al. (Sanger et al., 1977) revealed that the insert was identical with the 883 bp HO-1 sequence.